A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development
-
Published:2023-08-14
Issue:16
Volume:24
Page:12788
-
ISSN:1422-0067
-
Container-title:International Journal of Molecular Sciences
-
language:en
-
Short-container-title:IJMS
Author:
Bazzone Andre1ORCID, Barthmes Maria1, George Cecilia1, Brinkwirth Nina1, Zerlotti Rocco12, Prinz Valentin1, Cole Kim1, Friis Søren1, Dickson Alexander3, Rice Simon3, Lim Jongwon4ORCID, Fern Toh May4ORCID, Mohammadi Milad5, Pau Davide3, Stone David J.4, Renger John J.4, Fertig Niels1ORCID
Affiliation:
1. Nanion Technologies, Ganghoferstr. 70a, 80339 Munich, Germany 2. RIGeL-Regensburg International Graduate School of Life Sciences, University of Regensburg, 93053 Regensburg, Germany 3. SB Drug Discovery, West of Scotland Science Park, Glasgow G20 0XA, UK 4. Cerevel Therapeutics, 222 Jacobs St, Cambridge, MA 02141, USA 5. Assay Works, Am BioPark 11, 93053 Regensburg, Germany
Abstract
The lysosomal cation channel TMEM175 is a Parkinson’s disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.
Subject
Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis
Reference65 articles.
1. TMEM175 Is an Organelle K(+) Channel Regulating Lysosomal Function;Cang;Cell,2015 2. Structural basis for ion selectivity in TMEM175 K+ channels;Brunner;Elife,2020 3. The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture;Lee;Nature,2017 4. Oh, S., Paknejad, N., and Hite, R.K. (2020). Gating and selectivity mechanisms for the lysosomal K+ channel TMEM175. Elife, 9. 5. TMEM175 deficiency impairs lysosomal and mitochondrial function and increases α-synuclein aggregation;Jinn;Proc. Natl. Acad. Sci. USA,2017
Cited by
2 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献
|
|